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Abstract:

A novel method to produce hydrophobin monomers or multimers in plant
tissues is disclosed. A novel method to produce hydrophobin multimers in
E. coli cells is also described. The disclosure provides transgenic
plants, transgenic seeds, a production system, and expression cassettes
for making the transgenic plant carrying hydrophobin encoding gene
sequences.

6. The method of claim 1, wherein the hydrophobin sequence is expressed
as a monomer or as a multimer, and the multimer comprises several
hydrophobin polypeptide sequences separated from each other by a linker
sequence.

7. The method of claim 6, wherein the multimer is 3-12 mer.

8. The method of claim 7, wherein the linker sequence is a short
repetitive sequence from elastin, keratine, spider silk, silk, or
collagen proteins.

9. The method of claim 8, wherein the sequence is selected from the group
consisting of SEQ ID NO: 25 or SEQ ID NO:26.

10. An expression cassette for plant transformation, said cassette
comprising hydrophobin encoding sequence under control of a seed specific
promoter, a tuber specific promoter, a root specific promoter, an
inducible promoter or a constitutive plant promoter.

12. The expression cassette according to claim 10, wherein the
hydrophobin encoding sequence is encoding a hydrophobin monomer or a
hydrophobin multimer comprising several hydrophobin sequences separated
from each other by a linker sequence.

16. The transgenic seed of claim 15 wherein the seed is a Brassica seed
or a Camelina sativa seed.

17. A transgenic tuber comprising an expression cassette of claim 10.

18. A recombinant hydrophobin protein comprising 3-12 units of
hydrophobin polypeptides linked linearly to each other with a linker
sequence.

19. The recombinant hydrophobin protein of claim 18, wherein the linker
sequence is a short repetitive sequence isolated from the elastin,
spider-silk, silk, keratine or collagen.

20. The recombinant hydrophobin protein of claim 21, wherein the
hydrophobin units are essentially according to SEQ ID NO: 3.

Description:

CLAIM OF PRIORITY

[0001] This application claims priority of U.S. provisional application
No. 61/601,880 filed on Feb. 22, 2012 the contents of which are
incorporated herein by reference.

FIELD OF INVENTION

[0002] This invention relates to protein production and more specifically
to hydrophobin production and even more specifically to hydrophobin
production in plant tissue, preferably in seeds.

BACKGROUND OF THE INVENTION

[0003] Hydrophobins are small surface-active cystein-rich water-oil
soluble proteins characteristic of filamentous fungi. Hydrophobins can be
found in various fungal structures, such as areal hyphae, fruit bodies,
and spores. Hydrophobin encoding genes have been isolated from
ascomycetes, deuteromycetes and basidiomycetes. Some fungi have more than
one gene encoding hydrophobins. Currently more than 70 hydrophobin genes
have been isolated and characterized.

[0004] Hydrophobins have extraordinary character of reducing the surface
tension of water. Hydrophobins can render a hydrophobic surface to
hydrophilic and vice versa. Hydrophobins have a natural capability to
form membrane like structures; they are capable of making a one molecule
thick membrane between various phases.

[0005] Due to the extraordinary characters of hydrophobins, there is an
increasing need for these proteins in various industries. Hydrophobins
are useful in pharmaceutical and medical fields, in food- and chemical
industries, as well as in nanotechnologies. The ability of hydrophobins
to self assemble to membrane like structures is essential. Various
chemical and biochemical structures can be attached to hydrophobins to
create more complicated structures.

[0006] Industrial uses of hydrophobins have been described for example in:
US patent application publication 20110282229 for gluing paper products,
US patent application publication 20110268792 for use as excipients in
solid pharmaceutical formulations, US application publication 20110206820
in food industry, US application publication 20110086157 in use to form
oil-in-water emulsions, US patent application publication 20110017943 in
use to prevent ice formation on surfaces and in U.S. Pat. No. 7,393,448
for coating electrode surfaces.

[0007] Class I and class II hydrophobins, HFBI and HFBII respectively, are
known, each being about 100 amino acids long and having characteristic
hydropathy patterns. Almost all known hydrophobins contain eight
conserved cysteine residues that form intramolecular disulphide bridges.
Hydrophobins may be glycosylated, but their amino acid sequence is the
underlying cause of the amphipathic properties of hydrophobins.

In the past, the only way to obtain hydrophobins was to isolate them from
the natural fungus. However, the amounts of protein that can be obtained
using this method are inadequate for commercial applications. Due to the
increasing industrial need of hydrophobins there have been numerous
attempts to express and secrete recombinant hydrophobins in
micro-organisms. Harnessing the recombinant DNA technology for
hydrophobin production allows creation of hydrophobin variants that may
provide superior properties for use in specific applications. So far none
of the approaches have been satisfactory in terms of production yield:
the production levels are in the range of tens of milligrams in a litre
of growth medium, which is about 1/100- 1/1000 of the production levels
of commercial microbe cultures. An example of recombinant hydrophobin
expression is in patent application publication WO 00/058342, where the
endogenous HFBI hydrophobin was over expressed in Trichoderma reesei. The
production level was about 1.4 grams per liter of culture medium, which
is the highest production level achieved so far. However, for HFBII a
production level of only about 0.24 grams per liter has been shown. Other
production systems have been disclosed in US patent application
publication 2007/0077619, where Aspergillus nidulans hydrophobin was
expressed in Schizosaccharomyces pombe. US patent application publication
US 2009/0136996 discloses expression of hydrophobin in Escherichia coli.

[0008] Accordingly, production of hydrophobins in microbial cells has not
been satisfactory, partially because the achieved production levels are
not high enough for the industry needs. Partially the problem also lies
in the fact that hydrophobins are surfactants and therefore they cause
foaming of the culture medium. Thus the more the microbe secrets
hydrophobins, the more the culture medium will foam and the more
difficult it becomes to remove the foam from the fermentor. In US patent
US 2009/0136996 a solution to this problem has been offered by producing
hydrophobins in Escherichia coli--cells where the protein is accumulated
in the cell as inclusion bodies. The production levels however, are
extremely low: less than 10mg per liter of growth medium. Another problem
created by the E. coli--production system is that after the hydrophobins
are extracted from the cells their three dimensional structure has to be
restored.

[0009] Accordingly, there is a need for alternative production systems for
hydrophobin. Various industries need large amounts of hydrophobins
produced in efficient and economic methods. Hydrophobins are structural
proteins and therefore the amounts needed are considerably higher than
for proteins that act as enzymes. The current micro fermentation systems
do not satisfy the industrial needs. The instant invention provides an
alternative system that does not suffer from the deficiencies of the
current practices described above.

SUMMARY OF THE INVENTION

[0010] This invention provides solutions to the problems addressed above.
Accordingly, it is an object of the invention to provide a method to
produce recombinant hydrophobin protein in plant, said method comprising:
cloning a hydrophobin encoding sequence; constructing an expression
vector comprising the hydrophobin encoding sequence under a plant
promoter, transforming a plant cell with the expression vector; and
obtaining a transgenic plant expressing hydrophobin.

[0011] Another object of this invention is to provide an expression system
for producing multimeric hydrophobins.

[0012] Another object of the current invention is to provide an
alternative production system for the micro fermentation for hydrophobin
production.

[0013] Another object of the current invention is to provide an economic
method to produce high amounts of hydrophobins.

[0014] A further object of the current invention is to provide a method to
produce recombinant hydrophobins in plant cells.

[0015] Another object of the current invention is to provide a method to
produce recombinant hydrophobins in plant storage organs.

[0016] Yet another object of the current invention is to provide a method
to produce recombinant hydrophobins in plant seeds or tubers.

[0017] A further object of the current invention is to provide a method to
produce multimeric hydrophobins in E. coli production system.

[0018] A further object of the current invention is to provide a method to
produce recombinant hydrophobins in algal and cyanobacterial cells.

[0019] Another object of the current invention is to provide a method to
produce multimeric hydrophobins in plant production system.

[0020] An object of the current invention is to provide a method to
produce multimeric hydrophobin in plant storage organs.

[0021] Still another object of the current invention is to provide a
method to produce multimeric hydrophobins in plant seeds or tubers.

[0022] It is an object of this invention to provide expression vectors for
production of hydrophobins in plant cells.

[0023] It is a further object of this invention to provide expression
vectors for production of multimeric hydrophobins in plant cells.

[0024] It is yet another object of this invention to provide expression
vectors for production of hydrophobins in plant storage organs.

[0025] A further object of this invention is to provide expression vectors
for production of hydrophobins in plant seeds.

[0026] Another object of the current invention is to provide transgenic
plant cells expressing recombinant hydrophobin proteins.

[0027] Still another object of the current invention is to provide
transgenic plant seeds expressing recombinant hydrophobin proteins.

[0028] Yet another object of the current invention is to provide stably
transgenic plants expressing recombinant hydrophobin proteins.

[0029] Still another object of the current invention is to provide stably
transgenic plants expressing recombinant multimeric hydrophobin proteins.

[0030] Another object of this invention is recombinant hydrophobin protein
produced in plant cells.

[0031] Yet another object of this invention is a recombinant hydrophobin
multimer produced in plant cells.

[0032] A further object of this invention is a recombinant hydrophobin
protein produced in algal or cyanobacterial cells.

[0033] It is an object of this invention to provide a method to produce
recombinant hydrophobin protein in plant, where the method comprises
cloning a hydrophobin encoding sequence of fungal origin; constructing an
expression vector comprising the hydrophobin encoding sequence under a
plant promoter; transforming a plant cell with the expression vector; and
obtaining a transgenic plant expressing hydrophobin.

[0034] This invention also encompasses a method to produce recombinant
hydrophobin protein in algal or cyanobacterial cell, where the method
comprises the steps of cloning a hydrophobin encoding sequence;
constructing an expression vector comprising the hydrophobin encoding
sequence under an algal or cyanobacterial promoter, transforming an algal
or cyanobacterial cell with the expression vector; and obtaining a
transgenic alga or cyanobacterium expressing hydrophobin.

[0041] By cloning of hydrophobin encoding sequence is meant cloning of the
DNA sequence from a natural source, such as genomic DNA of a fungus or
using an artificially synthesized DNA molecule.

[0042] By monomeric hydrophobin is meant a single hydrophobin protein
unit.

[0043] By multimeric hydrophobin is meant a protein comprising more than
one hydrophobin unit linked one after another into a chain by linker
peptides.

[0044] By transient expression is meant a plant where the transgenic
material is introduced into the plant vegetative cells usually by
Agrofiltration.

[0045] By stably transformed plant is meant a transgenic plant where the
transgenic material is inserted into the genome of the plant, whereby the
transgenic character is inherited according to Mendelian rules.

[0046] To address the above described problems with currently existing
production systems this invention describes an E. coli production system
for expression of multimeric hydrophobins to increase the yield. In
another embodiment of this invention a plant production system for
monomeric and multimeric hydrophobins is described to increase the yield,
to eliminate the problems encountered by the micro fermentation systems
and to provide safe production system for a large scale production.

[0047] According to this invention hydrophobin is expressed in plant
tissue as a monomer or a multimer protein or alternatively as a multimer
in E. coli. According to this disclosure, plant tissue expression may be
transient, but preferably the system includes a stably transformed plant.
The multimeric hydrophobin is created by linking hydrophobin monomer
units to each other with an elastine-like linker peptides preferably
consisting of four amino acids and repeats there of. The linker peptides
could be selected from the group consisting of elastine, spider-silk,
silk, keratine and collagen. The length of the multimer chain can be
adjusted by changing the amount of hydrophobin monomers and the length of
the linker peptides. The number of the monomer units in the multimer
protein may be selected between 2 and 20, preferably between 3 and 12.

[0048] According to one preferred embodiment hydrophobin is expressed in
plant tissue as a hydrophobin-single chain antibody fusion protein. In
this embodiment hydrophobin may also be a monomer or a multimer with
linker peptides in between the hydrophobin units.

[0049] The current art include production of monomeric hydrophobin in E.
coli. The problems encountered by the known systems are low yield and
foaming of the culturemedium. One embodiment of the current disclosure
provides advantages that may minimize importance of these problems:
production of hydrophobin as a multimer in E. coli provides faster
organization of the protein to desired layers, as the hydrophobin-units
are already attached to each other in the multimeric form. Moreover, the
features of the recombinant protein may be easily modified by modifying
the number of hydrophobin-units and the length of the linkers in the
multimers.

[0050] Plant production systems have the advantage of producing large
amounts of proteins at low production cost. The need of hydrophobins is
great and the amounts needed are high because hydrophobin is not an
enzyme that acts as a catalyte. Hydrophobin is a structural protein and
the amounts needed are usually essentially higher than the current
micro-fermentation systems can provide. However, hydrophobins are
effective: few milligrams of hydrophobin is enough to cover about one
square meter with hydrophobin membrane. In solutions the required amounts
would be about 1/10000- 1/100 000 volume.

[0051] In food- and chemical industries there is a need for large amounts
of hydrophobins provided that the production costs remain low.

[0052] Because there are no human pathogens or bacterial toxins in the
plants, hydrophobin production in plant cells is safer than microbial
production. Importantly, hydrophobin produced in plant production system
is therefore safe for use in medical purposes. Plant production system
also enables production of hydrophobin-monomers or--multimers or fusion
proteins in large amounts in plant tissues.

[0053] Hydrophobin-encoding genes have never been stably transformed to
plant. However, Joensuu et al. (Plant Physiol. 2010 (152) pp. 622-633)
describe transient expression of hydrophobin as part of fusion protein,
where hydrophobin increases accumulation of the heterologous protein in
plant cells. Joensuu et al. show that the expression of Green Fluorescent
Protein (GFP) was increased markedly in the leaf tissue of tobacco plants
when the nucleic acid encoding GFP was linked with hydrophobin encoding
sequence. Joensuu et al. do not even suggest production of hydrophobins
in plant tissues. Importantly, the results of Joensuu et al. show that
while the fusion protein including hydrophobin was expressed in protein
bodies, the amount of RUBISCO-protein decreased to a level that would
suggest that the plant would not be able to live long. Based on the
information in Joensuu et al. one skilled in the art would not consider
stably transformed plant expressing hydrophobin to be a feasible solution
for the current problems encountered by the microfermentation production
systems. This disclosure provides a transgenic plant transformed with
construct where a nucleotide sequence encoding hydrophobin or
hydrophobin-multimer is linked with a plant promoter and terminator. It
is within the scope of this invention to provide transgenic plants of
various species. Preferably the transformed plants are dicotylenous
plants, but also monocotyledonous plants may be used. The transformed
plant may belong to the genus Brassicaceae. The transformed plant may be
oil seed rape, cauliflower, broccoli or Camelina sativa plant. Other
preferred dicotyledonous plant species include Nicotiana tabacum,
Medigago sativa, potato, sunflower, safflower, soybean, and alfalfa.
Preferred monocotyledonous plant species include corn, rice, barley and
duckweed (Lemna sp.). One skilled in the art will recognize that various
other plant species may be used as well. The production may also be
implemented by transforming algal and cyanobacterial cells with the
hydrophobin or hydrophobin-multimer encoding sequences.

[0054] Specifically, this disclosure provides constructs to express
hydrophobin in plant storage tissues and especially in the seeds.
Examples of such plant promoters and terminators directing the expression
to seeds are napin-promoter and terminator. Example of promoter directing
the expression to storage tissues is patatin-promoter. One skilled in the
art would recognize other promoters and terminators specific for the
target tissues. According to this disclosure, under non constitutive
promoters hydrophobin monomers or multimers can also be produced in the
green parts of the plant. Furthermore, hydrophobin production in storage
organs such as tubers and roots are within the scope of this invention.
Other plant organs, such as cauliflower heads may also be used for
hydrophobin expression.

[0055] The gene construct comprising hydrophobin encoding sequences is
transformed into the plant and the plant is grown until the leaves or
seeds emerge. The plant transformation may be carried out by
Agrobacterium-mediated transformation, but other methods, such as
particle bombardment or electroporation may as well be used. During the
development of the transgenic plant hydrophobin will accumulate depending
on the promoters into the leaves, storage tissues or in the seeds.
Hydrophobin expressed in the plant tissue is then extracted from the
storage organs, seeds or the leaves preferably by using two-phase method.

[0056] According to an alternative embodiment the expression of
hydrophobin and especially expression of multimeric hydrophobin may be
transient and the system comprises agroinfiltration.

[0057] The production level of hydrophobin in plant seeds is within a
range of 0.5-50 grams of hydrophobin per kilogram of seeds. To illustrate
the efficiency of this system a production level of 25 grams per kilogram
of rapeseed seeds would equal to about 50 kg of hydrophobin from a field
of one hectare or a greenhouse of 3000 square meters.

[0058] Hydrophobin produced in plant production system in greenhouse
facilities can be used in applications that require high quality
hydrophobin, such as medical and nanotechnological applications.
Hydrophobin production on the field conditions would be most practical
for purposes of food- and chemical industries where the required levels
are especially large.

[0059] The production of hydrophobin can also be harnessed into contained
facilities, by expressing hydrophobin encoding genes under inducible
promoters. One such method is described in U.S. Pat. No. 7,728,192.

[0060] Another embodiment of this invention is expression of multimeric
hydrophobin protein in procarytotic cells such as E. coli. So far E. coli
has been transformed with Trichoderma reesei's gene encoding monomeric
hydrophobin. One embodiment of this invention is described below, where
multimeric hydrophobin protein is expressed in E. coli.

[0061] The invention is now described by means of non-limiting examples.
One skilled in the art would understand that several changes can be made
to the method without diverting from the spirit of the invention. It is
understood by one skilled in the art, that even if the examples describe
vectors containing Schitsophyllum commune hydrophobin encoding gene any
one of the various known hydrophobin encoding sequences may as well be
used. Similarly, while the examples describe Camelina sativa as the
target plant, other species may as well be used.

[0065] Alternatively, detection- and purification tags like 6HIS- or
STREP-tags can be introduced to C-terminus of each hydrophobin by using a
longer 3' primers containing coding sequence of desired tag.

[0068] Alternatively, detection- and purification tags, like 6HIS- or
STREP-tags, can be introduced to C-terminus of hydrophobin by using a
longer 3'primer sequence containing coding sequence of desired tag.

[0073] The basic transformation protocol for Camelina sativa was applied
with all the hydrophobin and hydrophobin derivatives vectors.

[0074] The seeds of Camelina sativa plant grown in greenhouse are
sterilized by immersing in 70% ethanol for 1 min and then treating for 5
min with Na-hypochlorite solution (2% active CL) with an addition of
Tween-20 (1 drop per 100 ml). After sterilization the seeds are washed
four times in sterile water and placed on solid Murashige and Skoog (MS)
agar medium (Murashige and Skoog, Physiol. Plant. 15:472-493, 1962)
without sugars for germination. Sterilized seeds are germinated and grown
2 weeks on solid Murashige and Skoog (MS) medium without hormones First
true leaves serve as a source of explants for transformation procedure.

[0077] All the Murashige and Skoog (MS) culture media are supplemented
with 1% sucrose and all in vitro cultures are kept at temperatures of
25° C. (day) and 18° C. (night) under the photoperiod of 16
h.

[0078] The explants are washed with water containing 300 mg/l ticarcillin.
The explants are placed on Murashige and Skoog (MS) medium supplemented
with hormones [0.7 mg/l 6-benzylaminopurine (BAP), 0.3 mg/l
α-naphthaleneacetic acid (NAA)] and 200 mg/l ticarcillin and 0.05
mg/L imidazole. Two to three weeks old shoots are placed to the normal or
half strength Murashige and Skoog (MS) medium solidified with 0.7% agar
and supplemented with 200 mg/l Ticarcillin and optionally 0,1 mg/L
imidazole and 0.3 mg/l α-naphthalene acetic acid (NAA). Rooted
shoots are transferred to soil and transgenic plants are grown in
greenhouse or comparable conditions. Transgenic plants are tested for
recombinant gene expression with a northern blot analysis and the
presence of the transgene is confirmed with Southern analysis.

[0081] 400 mg of Triton X-114 was weighed into the 15 ml test tube. 10 ml
of leaf extract was preheated in 24° C. water bath for 5 min and
leaf extract was pipeted to a test tube containing Triton X-114. The leaf
extract/Triton X-114 mixture was vortexed thoroughly for 1 min. The tube
was set back to 24° C. water bath for 20 min to separate phases.

[0082] The lower phase containing hydrophobin was transferred by pipeting,
starting from the bottom of the tube, to a new tube. 4 ml isobutanol was
added to hydrophobin containing lower phase and mixed well. The tube was
set to a 24° C. water bath to separate phases. The lower phase
containing the hydrophobin was collected to a new test tube. Isobutanol
was removed by filtration through Biogel P-6 column (Bio-Rad).
Hydrophobin containing solution was stored at 4° C. temperature.

Seeds of Camelina sativa

[0083] 0.5 g of freshly harvested Camelina sativa seeds expressing
hydrophobin or hydrophobin multimer were put into mortar and covered with
liquid nitrogen. Seeds were grind to fine powder. Mortar was set on ice
and the powder was let to thaw and 15 ml PBS (Phosphate-buffered saline
extraction buffer: 137 mM NaCl, 2.7M KCl, 8.1 mM Na2HPO4 and
1.8 mM KH2PO4; pH7.4 was added. Extract was transferred to test tube and
centrifuged 5 min at 20 000 at 4 ° C. The supernatant was
collected into the new tube.

[0084] 400 mg of Triton X-114 was weighed into the 15 ml test tube. 10 ml
of leaf extract was preheated in 24° C. water bath for 5 min and
leaf extract was pipeted to a test tube containing Triton X-114. The leaf
extract/Triton X-114 mixture was vortexed thoroughly for 1 min. The tube
was set back to 24° C. water bath for 20 min to separate phases.

[0085] The lower phase containing hydrophobin was transferred by pipeting,
starting from the bottom of the tube, to a new tube. 4 ml isobutanol was
added to hydrophobin containing lower phase and mixed well. The tube was
set to a 24° C. water bath to separate phases. The lower phase
containing the hydrophobin was collect to a new test tube. Isobutanol was
removed by filtration through Biogel P-6 column (Bio-Rad). Hydrophobin
containing solution was stored at 4° C. temperature.

EXAMPLE 7

SDS-PAGE--and Southern Blot Analyses

[0086] SDS-PAGE Analysis

[0087] 75 μl sample of hydrophobin containing solution prepared
according to example 6 is mixed with 25 μl of 4X SDS-PAGE loading
buffer. The sample is boiled for 5 min in water bath. 20 μl of the
sample is loaded on well. The gel is run 200 V until the dye front
reaches the end of the gel. The gel is washed twice for 15 min with
water. The gel is stained according to manufacturer instructions over
night (GelCode Blue Stain Reagent). The gGel is destained with water for
1 hour.

Southern Blot Analysis

[0088] Total genomic DNA is isolated from leaf tissue of transgenic
Camelina sativa plants using DNeasy Plant Midi Kit according to the
supplier's instructions (Qiagen). Three μg of DNA from hydrophobin
positive Camelina sativa plants is digested with appropriate restriction
enzymes, cutting out a suitable fragment with described hydrophobin- or
hydrophobin derivative gene expression cassette from the T-region of
recombinant DNA inserted in the plant genome.

[0089] Digested DNA samples are separated in a 0.7% agarose (Promega) gel
overnight at 15 mA current and transferred to positively charged nylon
membrane (Boehringer Mannheim) using vacuum blotter. RNA probes are
synthesized using T3 RNA polymerase on the pBluescript vector carrying
corresponding hydrophobin gene sequence and labeled with
digoxigenin-11-UTP. The membrane is hybridized and developed according to
the supplier's instructions (Boehringer Mannheim, The DIG user's guide
for filter hybridization). The membrane is prehybridized at 50° C.
for 2 h and hybridized at 50° C. in a "DIG Easy Hyb" hybridization
solution (Boehringer Mannheim) overnight with a digoxigenin-UTP labeled
RNA probe. The concentration of RNA probe is 100 ng/ml.

[0090] After hybridization the membrane is washed in SSC buffers, blocked
and detected using Anti-Digoxigenin-AP alkaline phosphatase substrate
(Boehringer Mannheim). Chemiluminescent detection is done with
CSPD-substrate and the membrane is exposed to X-ray film (Boehringer
Mannheim). Presence of the transgene insertion is proved in comparison to
DNA of non-transgenic Camelina sativa plant DNA as negative control, and
to plasmid DNA carrying the gene sequence mixed with non-transgenic plant
DNA as positive control.

[0092] The production of multimeric hydrophobin is exemplified in commonly
used microbial expression system, by production in Escherichia coli. pOne
skilled in the art would realize that other microbial systems may also be
used.

[0093] Multimeric hydrophobin gene is amplified from plant transformation
vector for polymeric hydrophobin (example 3), with specific primer pair
excluding the signal peptide. For generating a blunt-end PCR product, PCR
is carried out with Finnzymes (Thermo scientific) Phusion high-fidelity
PCR polymerase, with proofreading activity. Reaction is set up according
to manufacturer's instructions. PCR product is run in 0.8% agarose gel
with EtBr as routinely, purified and cloned into pET101/D-TOPO expression
vector according to commonly known procedure. Cloning reaction is
transformed to E. coli TOP10 cells, positive colonies are screened
according to standard protocols, positive transformants are selected and
plasmid DNA is isolated from 2-4 positive clones. Plasmid is sent for
sequencing, to verify that no mutations are generated by PCR and cloning
procedure.

[0094] Once the plasmid sequence is verified, selected plasmid is
transformed to BL21-AI E. coli cells. Positive clones are screened and
several of them are selected for expression of the multimeric target
protein. Expression was induced by culturing the cells to early
exponential growth phase and by adding Isopropyl
f3-D-1-thiogalactopyranoside (IPTG) to 1 mM concentration. Appropriate
induction time is set by taking samples from different time points during
several hours of induction.